Activation of the multicomponent antigen receptor in T cells (TCR) results in rapid activation of a protein tyrosine kinase pathway. To fully understand the function of this pathway, the kinase and its substrates must be characterized. Two substrates for the tyrosine phosphorylation pathway are the TCR-zeta chain and the gamma form of phospholipase C. To identify other substrates we have used antiphosphotyrosine antibodies to isolate sufficient quantities of two additional substrates for partial amino acid sequence determination. The identity of an 81kD substrate is ezrin, a well characterized substrate of the growth factor receptor tyrosine kinases. Partial sequence of a 100kD substrate enabled us to clone the corresponding cDNA. The protein has sequence homology with several yeast proteins involved in intracellular vesicular function. Anti-peptide antibodies immunoblot and immunoprecipitate the tyrosine phosphorylated 100kD substrate in B cells and mast cells as well as T cells. Using digitonin as a detergent to solubilize a murine T cell, we have demonstrated that the protein tyrosine kinase (fyn) is non-covalently associated with the TCR. Twenty-25% of intracellular (fyn) is associated with the TCR; conversely, only 2-5% of TCR is associated with the kinase. The association has also been demonstrated using chemical crosslinkers introduced into T cells via a permeabilization protocol. This technique also allows demonstration of an association between the TCR-fyn complex, the membrane tyrosine phosphatase, CD45, and the stimulatory molecule, Thy 1.